One of the greatest challenges of developing post-exposure treatments for botulism caused by clostridial neurotoxins is the brief therapeutic window for conventional antibody-based antitoxins to neutralize the free neurotoxin in the circulation and to block binding of the toxin to cellular receptors. Because of this, the patient is most often asymptomatic until after the toxin has already been internalized and is no longer accessible to antibody neutralization. No current antitoxin is effective after toxin has been internalized and symptoms manifested. Our proposed post-exposure antitoxin will address this critical need. We propose to develop a post-exposure therapeutic anti-botulinum neurotoxin (BoNT) reagent, which builds on the concept of an antibody/inhibitor-toxin chimera that can be targeted for specific delivery into neuronal cells and that upon entry into the cells will deliver and release an inhibitor into the cytosol that will neutralize the catalytic activity of BoNT, such that the cells can more rapidly recover. In this chimeric antitoxin, high affinity scFv antibodies, peptide-based inhibitors, or small molecule-based inhibitors will be attached to the heavy chain of BoNT (BoNT-HC), such that it could specifically inhibit the metalloprotease activity of BoNT-LC after specific receptor binding, translocation (i.e. delivery), and release into the cytosol of neuronal cells. The advantage of using this system is that the neutralizing antitoxin will be directed to the same location where toxin action is occurring - not only the same type of cells, but also the same intracellular compartments where the target substrates are located. Importantly, our proposed antitoxin strategy has the potential to reverse and/or shorten the duration of the clinical effects of intoxication (i.e. paralysis). To achieve the goal of obtaining this new post-exposure antitoxin therapeutic, we propose the following aims: 1. Construction and optimization of recombinant BoNT heavy chain fusion proteins as delivery vehicles. 2. Optimization of the cargo release mechanism for efficient delivery of inhibitor to the cytosol of neuronal cells. 3. Optimization of the substrate-scFv inhibitor as a scaffold for neutralization of BoNT activity. 4. Construction and optimization of alternative BoNT/A-HC adaptors for delivering peptide-based and small-molecule-based inhibitors. [unreadable] [unreadable] [unreadable]